Nucleotide sequences which code for the pck gene

ABSTRACT

The invention relates to isolated nucleotide sequences from Coryneform bacteria which code for the pck gene encoding the enzyme phosphoenol pyruvate carboxykinase (PEP carboxykinase). The invention also relates a process for the fermentative preparation of L-amino acids, in particular L-lysine, L-threonine, and L-glutamate by attenuation of the pck gene.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of U.S. Pat. application Ser. No. 10/059,091 filed Jan. 30, 2002, now abandoned, which is a divisional of U.S. patent appl. Ser. No. 09/455,777, filed Dec. 7, 1999, now U.S. Pat. No. 6,420,151, which claims priority to German Patent Application No. 19950409.1 filed on Oct. 20, 1999.

FIELD OF THE INVENTION

The invention provides nucleotide sequences from coryneform bacteria which code for the pck gene and a process for the fermentative preparation of L-amino acids, in particular L-lysine, L-threonine, and L-glutamate by attenuation of the pck gene.

DESCRIPTION OF BACKGROUND ART

Amino acids, in particular lysine and threonine, are used in animal nutrition, in the foodstuffs industry, in the pharmaceuticals industry and in human medicine.

It is known that these substances are prepared by fermentation of strains of coryneform bacteria, in particular Corynebacterium glutamicum. Because of its great importance, work is constantly being undertaken to improve the preparation processes. Improvements to the processes can relate to fermentation measures, such as e.g. stirring and supply of oxygen, or the composition of the nutrient media, such as e.g. the sugar concentration during the fermentation, or the working up to the product form by e.g. ion exchange chromatography, or the intrinsic output properties of the microorganism itself.

Methods of mutagenesis, selection and mutant selection are used to improve the output properties of these microorganisms. Strains which are resistant to antimetabolites or are auxotrophic for metabolism products of regulatory importance and produce the desired amino acid are obtained in this manner.

Methods of the recombinant DNA technique have also been employed for some years for improving the strain of Corynebacterium strains which produce L-amino acid.

SUMMARY OF THE INVENTION

The invention provides an isolated polynucleotide from coryneform bacteria, comprising a polynucleotide sequence chosen from the group consisting of

-   a) polynucleotide which is identical to the extent of at least 70%     to a polynucleotide which codes for a polypeptide which comprises     the amino acid sequence of SEQ ID No. 2, -   b) polynucleotide which is identical to the extent of at least 70%     to a polynucleotide which codes for the polypeptide mentioned and is     contained on the plasmid pEK-pckA (FIG. 1) or pEK-pckB (FIG. 2), -   c) polynucleotide which codes for a polypeptide which comprises an     amino acid sequence which is identical to the extent of at least 70%     to the amino acid sequence of SEQ ID No. 2, -   d) polynucleotide which is complementary to the polynucleotides of     a), b) or c), -   the polypeptide preferably having the enzymatic activity of the     phosphoenol pyruvate carboxykinase (PEP carboxykinase).

The invention also provides a preferably recombinant DNA with Corynebacterium origin which is capable of replication in coryneform microorganisms and contains at least the nucleotide sequence which codes for the pck gene, shown in SEQ ID No. 1.

The invention also provides a DNA according to claim 1 which is capable of replication, comprising

-   (i) the nucleotide sequence shown in SEQ ID no. 1, or -   (ii) at least one sequence which corresponds to sequence (i) within     the range of the degeneration of the genetic code, or -   (iii) at least one sequence which hybridizes with the sequence     complementary to sequence (i) or (ii), and/or optionally -   (iv) sense mutations of neutral function in (i) which do not modify     the enzymatic activity of the protein/polypeptide.

The invention also provides

-   a polynucleotide according to claim 2, comprising the nucleotide     sequence as shown in SEQ ID no. 1, -   a polynucleotide according to claim 2, which codes for a polypeptide     which comprises the amino acid sequence as shown in SEQ ID No. 2, -   a vector containing the polynucleotide according to claim 1, in     particular pEK-pckA or pEK-pckB, shown in FIGS. 1 and 2, -   polynucleotides comprising at least 15 successive nucleotides chosen     from the nucleotide sequence of SEQ ID No. 1 between positions 1 and     728, -   polynucleotides comprising at least 15 successive nucleotides chosen     from the nucleotide sequence of SEQ ID No. 1 between positions 1103     and 2021, -   polynucleotides comprising at least 15 successive nucleotides chosen     from the nucleotide sequence of SEQ ID No. 1 between positions 2022     and 3854, -   polynucleotides comprising at least 15 successive nucleotides chosen     from the nucleotide sequence of SEQ ID No. 1 between positions 3885     and 3935, -   and coryneform bacteria serving as the host cell, into which the     Δpck deletion has been incorporated.

The invention also provides polynucleotides which substantially comprise a polynucleotide sequence, which are obtainable by screening by means of hybridization of a corresponding gene library, which comprise the complete gene with the polynucleotide sequence corresponding to SEQ ID no. 1, with a probe which comprises the sequence of the polynucleotide mentioned, according to SEQ ID no. 1 or a fragment thereof, and isolation of the DNA sequence mentioned.

Polynucleotide sequences according to the invention are suitable as hybridization probes for RNA, cDNA and DNA, in order to isolate, in the full length, cDNA which code for phosphoenol pyruvate carboxykinase and to isolate those cDNA or genes which have a high similarity of sequence with that of the phosphoenol pyruvate carboxykinase gene.

They are also suitable for incorporation into so-called “arrays” or “DNA chips” in order to detect and determine the corresponding polynucleotides, e.g., m-RNA.

Polynucleotide sequences according to the invention are furthermore suitable as primers with the aid of which DNA of genes which code for the phosphoenol pyruvate carboxykinase can be prepared with the polymerase chain reaction (PCR).

Such oligonucleotides which serve as probes or primers comprise at least 25, 26, 27, 28, 29, or 30, with a maximum length of 35 or 40, preferably at least 20, 21, 22, 23, or 24, with a maximum length of 30 or 35, very particularly at least 15, 16, 17, 18, or 19, with a maximum length of 25 or 30, successive nucleotides. Oligonucleotides with a length of at least 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40, with a maximum length of 45 or 50, or at least 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50, with a maximum length of 55 or 60 nucleotides are also suitable. Oligonucleotides with a length of at least 100, 150, 200, 250 or 300 nucleotides with a maximum length of 350, 400, or 500 are optionally also suitable.

“Isolated” means separated out of its natural environment.

“Polynucleotide” in general relates to polyribonucleotides and polydeoxyribonucleotides, it being possible for these to be non modified RNA or DNA or modified RNA or DNA.

The polynucleotides according to the invention include a polynucleotide according to SEQ ID No. 2 or a fragment prepared therefrom and also to those which are at least in particular 70% to 80%, preferably at least 81% to 85%, particularly preferably at least 86% to 90%, and very particularly preferably at least 91%, 93%, 95%, 97% or 99% identical to the polynucleotide according to SEQ ID No. 1 or a fragment prepared therefrom.

“Polypeptides” are understood as meaning peptides or proteins which comprise two or more amino acids bonded via peptide bonds.

The polypeptides according to the invention include a polypeptide according to SEQ. ID No.:2, in particular those with the biological activity of phosphoenol pyruvate carboxykinase and also those which are at least 70% to 80%, preferably at least. 81% to 85%, particularly preferably at least 86% to 90%, and very particularly preferably at least 91%, 93%, 95%, 97% or 99% identical to the polypeptide according to SEQ ID No:2 and have the activity mentioned.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention will be described with reference to the drawings, in which the stated base pair numbers are approximate values obtained in the context of reproducibility, and in which:

FIG. 1 is a restriction map of the plasmid pEK-pckA;

FIG. 2 is a restriction map of the plasmid pEK-pckB; and

FIG. 3 is a restriction map of the plasmid pk19mobsacBΔpck.

DETAILED DESCRIPTION

Amino acids, in particular L-lysine and L-threonine, are used in animal nutrition, in the foodstuffs industry, in the pharmaceuticals industry and in human medicine. There is therefore a general interest in providing new improved processes for the preparation of these products.

Polynucleotide sequences according to the invention are suitable as hybridization probes for RNA, cDNA and DNA, in order to isolate, in the full length, cDNA which code for phosphoenol pyruvate carboxykinase and to isolate those cDNA or genes which have a high similarity of sequence with that of the phosphoenol pyruvate carboxykinase gene.

The invention also provides a process for the fermentative preparation of L-amino acids, in particular L-lysine and L-threonine, using coryneform bacteria which in particular already produce the L-amino acids and in which the nucleotide sequence(s) which code(s) for the pck gene are attenuated, in particular expressed at a low level.

The term “attenuation” in this connection describes the reduction or elimination of the intracellular activity of one or more enzymes (proteins) in a microorganism which are coded by the corresponding DNA, for example by using a weak promoter or using a gene or allele which codes for a corresponding enzyme with a low activity or inactivates the corresponding enzyme (protein), and optionally combining these measures.

The microorganisms which the present invention provides can produce L-amino acids, in particular lysine and threonine, from glucose, sucrose, lactose, fructose, maltose, molasses, starch, cellulose or from glycerol and ethanol. They can be representatives of coryneform bacteria, in particular of the genus Corynebacterium. Of the genus Corynebacterium, there may be mentioned in particular the species Corynebacterium glutamicum, which is known among specialists for its ability to produce L-amino acids.

Suitable strains of the genus Corynebacterium, in particular of the species Corynebacterium glutamicum, are, for example, the known wild-type strains

Corynebacterium glutamicum ATCC13032

Corynebacterium acetoglutamicum ATCC15806

Corynebacterium acetoacidophilum ATCC13870

Corynebacterium thermoaminogenes FERM BP-1539

Corynebacterium melassecola ATCC17965

Brevibacterium flavum ATCC14067

Brevibacterium lactofermentum ATCC13869 and

Brevibacterium divaricatum ATCC14020

and L-amino acid-producing mutants or strains prepared therefrom,

such as, for example, the lysine-producing strains

Corynebacterium glutamicum FERM-P 1709

Brevibacterium flavum FERM-P 1708

Brevibacterium lactofermentum FERM-P 1712

Corynebacterium glutamicum FERM-P 6463

Corynebacterium glutamicum FERM-P 6464 and

Corynebacterium glutamicum DSM5714 or

such as, for example, the L-threonine-producing strains

Corynebacterium glutamicum ATCC21649

Brevibacterium flavum BB69

Brevibacterium flavum DSM5399

Brevibacterium lactofermentum FERM-BP 269

Brevibacterium lactofermentum TBB-10

Corynebacterium glutamicum MH20-22B-DR17.

The inventors have succeeded in isolating the new pck gene of C. glutamicum which codes for the enzyme phosphoenol pyruvate carboxykinase (PEP carboxykinase) (EC 4.1.1.49).

To isolate the pck gene or also other genes of C. glutamicum, a gene library of this microorganism is first set up in E. coli. The setting up of gene libraries is described in generally known textbooks and handbooks. The textbook by Winnacker: Gene und Klone, Eine Einführung in die Gentechnologie [Genes and Clones, An Introduction to Genetic Engineering] (Verlag Chemie, Weinheim, Germany, 1990) or the handbook by Sambrook et al.: Molecular Cloning, A Laboratory Manual (Cold Spring Harbor Laboratory Press, 1989) may be mentioned as an example. A well-known gene library is that of the E. coli K-12 strain W3110 set up in λ vectors by Kohara et al. (Cell 50, 495-508 (1987)). Bathe et al. (Molecular and General Genetics, 252:255-265, 1996) describe a gene library of C. glutamicum ATCC13032, which was set up with the aid of the cosmid vector SuperCos I (Wahl et al., 1987, Proceedings of the National Academy of Sciences USA, 84:2160-2164) in the E. coli K-12 strain NM554 (Raleigh et al., 1988, Nucleic Acids Research 16:1563-1575). Börmann et al. (Molecular Microbiology 6(3), 317-326)) in turn describe a gene library of C. glutamicum ATCC13032 using the cosmid pHC79 (Hohn and Collins, Gene 11, 291-298 (1980)).

To prepare a gene library of C. glutamicum in E. coli, it is also possible to use plasmids or plasmid vectors, such as, for example, pBR322 (Bolivar, Life Sciences, 25, 807-818 (1979)), pUC9 (Vieira et al., 1982, Gene, 19:259-268), pACYC177 (Chang and Cohen, Journal of Bacteriology 134, 1141-1156 (1978)) or pSC101 (Cohen and Chang, Journal of Bacteriology 132, 734-737 (1977)). Suitable hosts are, in particular, those E. coli strains which are restriction- and recombination-defective.

The gene library is then incorporated into an indicator strain by transformation (Hanahan, Journal of Molecular Biology 166, 557-580, 1983) or electroporation (Tauch et.al., 1994, FEMS Microbiological Letters, 123:343-347). The indicator strain is distinguished in that it has a mutation in the gene of interest which causes a detectable phenotype. In the context of the present invention, the E. coli mutant HG4 described by Goldie and Sanwal (Journal of Bacteriology 141: 1115-1121 (1980)) is of importance. This strain carried a mutation in the pck gene, as a result of which growth on succinate as the sole source of carbon is severely impaired. By transformation with a vector which contains the pck gene, growth on succinate can be re-established.

The long DNA fragments cloned with the aid of cosmids or other vectors can than be subcloned into known plasmid vectors in the form of shorter DNA fragments. Assignment of the gene according to the invention to a specific DNA section is made possible as a result. For this, plasmid vectors known from the prior art, such as e.g. pBR322 (Bolivar, Life Sciences, 25, 807-818 (1979)) or the pSU vectors described by Bartolomé et al. (Gene 102, 75-78 (1991)) are used. However, shuttle vectors which replicate both in Escherichia coli and in Corynebacterium glutamicum, such as e.g. pZ1 (Menkel et al., Applied and Environmental Microbiology (1989) 64: 549-554) or pEK0 (Eikmanns et al., Gene 102 (1991)) are preferably used, so that investigations can be carried out in both species. Examples of these are the plasmids pEK-pckA (FIG. 1) and pEK-pckB (FIG. 2), which were prepared starting from the plasmid vector pEK0 and carry the pck gene according to the invention.

The DNA sections characterized in this manner are then again subcloned into the usual vectors suitable for DNA sequencing. Alternatively, the long DNA sections cloned in cosmids can be subcloned directly into sequencing vectors. Examples of such vectors which are suitable for DNA sequencing are the plasmids pGEM-5zf(−) or pGEM-5zf(+) from the company Promega Corporation (Promega Protocols and Application Guide, Second Edition, 1991, part number Y981, Promega Corporation, Madison, Wis., USA).

Methods of DNA sequencing are described, inter alia, by Sanger et al. (Proceedings of the National of Sciences of the United States of America USA, 74:5463-5467, 1977).

The DNA sequences obtained can then be investigated with known algorithms or sequence analysis programs, such as e.g. that of Staden (Nucleic Acids Research 14, 217-232(1986)), the GCG program of Butler (Methods of Biochemical Analysis 39, 74-97 (1998)) the FASTA algorithm of Pearson and Lipman (Proceedings of the National Academy of Sciences USA 85,2444-2448 (1988)) or the BLAST algorithm of Altschul et al. (Nature Genetics 6, 119-129 (1994)) and compared with the sequence entries which exist in databanks accessible to the public. Databanks for nucleotide sequences which are accessible to the public are, for example, that of the European Molecular Biologies Laboratories (EMBL, Heidelberg, Germany) of that of the

National Center for Biotechnology Information (NCBI, Bethesda, Md., USA).

The new DNA sequence of C. glutamicum which codes for the pck gene and which, as SEQ ID No. 1, is a constituent of the present invention was obtained in this manner. The amino acid sequence of the corresponding protein has furthermore been derived from the present DNA sequence by the methods described above. The resulting amino acid sequence of the pck gene product is shown in SEQ ID No. 2. Endogenous enzymes are able to cleave the N-terminal amino acid methionine or formylmethionine of the protein.

Coding DNA sequences which result from SEQ ID No. 1 by the degeneracy of the genetic code are also a constituent of the invention.

In the same way, DNA sequences which hybridize with SEQ ID No. 1 or parts of SEQ ID No. 1 are a constituent of the invention. Conservative amino acid exchanges, such as e.g., exchange of glycine for alanine or aspartic acid for glutamic acid in proteins are furthermore known among experts as “sense mutations” which do not lead to a fundamental change in the activity of the protein, i.e., one of neutral function. It is furthermore known that changes on the N and C terminus of a protein cannot substantially impair or can even stabilize the function thereof. Information in this context can be found by the expert, inter alia, in Ben-Bassat et al. (Journal of Bacteriology 169:751-757 (1987)), in O'Regan et al. (Gene 77:237-251 (1989)), in Sahin-Toth et al. (Protein Sciences 3:240-247 (1994)), in Hochuli et al. (Bio/Technology 6:1321-1325 (1988)) and in known textbooks of genetics and molecular biology. Amino acid sequences which result in a corresponding manner from SEQ ID No. 2 are also a constituent of the invention.

In the same way, DNA sequences which hybridize with SEQ ID No. 1 are a constituent of the invention. Finally, DNA sequences which are prepared by the polymerase chain reaction (PCR) using primers which result from SEQ ID No. 1 are a constituent of the invention. Such oligonucleotides typically have a length of at least 15 nucleotides.

Instructions for identifying DNA sequences by means of hybridization can be found by the expert, inter alia, in the handbook “The DIG System Users Guide for Filter Hybridization” from Boehringer Mannheim GmbH (Mannheim, Germany, 1993) and in Liebl et al. (International Journal of Systematic Bacteriology (1991) 41:255-260). The hybridization takes place under stringent conditions, that is to say only hybrids in which the probe and target sequence, i.e., the polynucleotides treated with the probe, are at least 70% identical and formed. It is known that the stringency of the hybridization, including the washing steps, is influenced or determined by varying the buffer composition, the temperature and the salt concentration. The hybridization reaction is preferably carried out under a relatively low stringency compared with the washing steps (Hybaid Hybridisation Guide, Hybaid Limited, Teddington, UK, 1996).

A b×SSC buffer at a temperature of approximately 50-60° C., for example, can be employed for the hybridization reaction. Probes can also hybridize here with polynucleotides which are less than 70% identical to the sequence of the probe. Such hybrids are less stable and are removed by washing under stringent conditions. This can be achieved, for example, by lowering the salt concentration to 2×SSC and subsequently to 0.5×SSC (The DIG System User's Guide for Filter Hybridization, Boehringer Mannheim, Mannheim, Germany, 1995) a temperature of approximately 50-68° C. being established. It is optionally possible to lower the salt concentration to 0.1×SSC. Polynucleotide fragments which are, for example, at least 70% or at least 80% or at least 90% to 95% identical to the sequence of the probe employed can be isolated by increasing the hybridization temperature stepwise in steps of approximately 1-2° C. Further instructions on hybridization are obtainable on the market in the form of so-called kits (e.g., DIG Easy Hyb from Roche Diagnostics GmbH, Mannheim, Germany, Catalogue No. 1603558).

The inventors have found that coryneform bacteria produce L-amino acids, in particular lysine and threonine, in an improved manner after attenuation of the pck gene.

To achieve an attenuation, either the expression of the pck gene or the catalytic properties of the enzyme protein can be reduced or eliminated. The two measures can optionally be combined.

The reduction in gene expression can take place by suitable culturing or by genetic modification (mutation) of the signal structures of gene expression. Signal structures of gene expression are, for example, repressor genes, activator genes, operators, promoters, attenuators, ribosome binding sites, the start codon and terminators. The expert can find information on this e.g. in the patent application WO 96/15246, in Boyd and Murphy (Journal of Bacteriology 170: 5949 (1988)), in Voskuil and Chambliss (Nucleic Acids Research 26: 3548 (1998), in Jensen and Hammer (Biotechnology and Bioengineering 58: 191 (1998)), in Patek et al. (Microbiology 142: 1297 (1996) and in known textbooks of genetics and molecular biology, such as e.g. the texbook by Knippers (“Molekulare Genetik [Molecular Genetics]”, 6th edition, Georg Thieme Verlag, Stuttgart, Germany, 1995) or that by Winnacker (“Gene und Klone [Genes and Clones”, VCH Verlagsgesellschaft, Weinheim, Germany, 1990).

Mutations which lead to a change or reduction in the catalytic properties of enzyme proteins are known from the prior art; examples which may be mentioned are the works by Qiu and Goodman (Journal of Biological Chemistry 272: 8611-8617 (1997)), Sugimoto et al. (Bioscience Biotechnology and Biochemistry 61: 1760-1762 (1997)) and Möckel (“Die Threonindehydratase aus Corynebacterium glutamicum: Aufhebung der allosterischen Regulation und Struktur des Enzyms [Threonine dehydratase from Corynebacterium glutamicum: Cancelling the allosteric regulation and structure of the enzyme]”, Reports from the Julich Research Centre, Jül-2906, ISSN09442952, Jülich, Germany, 1994). Comprehensive description can be found in known textbooks of genetics and molecular biology, such as e.g. that by Hagemann (“Allgemeine Genetik [General Genetics]”, Gustav Fischer Verlag, Stuttgart, 1986).

Possible mutations are transitions, transversions, insertions and deletions. Depending on the effect of the amino acid exchange on the enzyme activity, missense mutations or nonsense mutations are referred to. Insertions or deletions of at least one base pair in a gene lead to frame shift mutations, which lead to incorrect amino acids being incorporated or translation being interrupted prematurely. Deletions of several codons typically lead to a complete loss of the enzyme activity. Instructions on generation of such mutations are prior art and can be found in known textbooks of genetics and molecular biology, such as e.g. the textbook by Knippers (“Molekulare Genetik [Molecular Genetics]”, 6th edition, Georg Thieme Verlag, Stuttgart, Germany, 1995), that by Winnacker (“Gene und Klone [Genes and Clones]”, VCH Verlagsgesellschaft, Weinheim, Germany, 1990) or that by Hagemann (“Allgemeine Genetik [General Genetics]”, Gustav Fischer Verlag, Stuttgart, 1986).

An example of a mutated pck gene is the Δpck allele contained in the plasmid pK19mobsacBΔpck (FIG. 3). The Δpck allele contains only the 5′ and the 3′ flanks of the pck gene; a section of the coding region 1071 bp long is missing (deletion). This Δpck allele can be incorporated into coryneform bacteria by integration mutagenesis. The abovementioned plasmid pK19mobsacBΔpck, which is not capable of replication in C. glutamicum, is used for this. After transfer by conjugation or transformation and homologous recombination by means of a first “cross-over” event which effects integration and a second “cross-over” event which effects excision in the pck gene, the incorporation of the Δpck allele is achieved and a total loss of the enzyme function in the particular strain is achieved.

Instructions and explanations on integration mutagenesis are to be found, for example, in Schwarzer and Pühler (Bio/Technology 9,84-87 (1991)) or Peters-Wendisch et al. (Microbiology 144, 915-927 (1998)).

Examples of amino acid-producing strains of coryneforme bacteria with an attenuated pck gene are the lysine-producing strain MH20-22BΔpck and the threonine-producing strain DM368-2Δpck.

In addition, it may be advantageous for the production of L-amino acids to over-express one or more enzymes of the particular biosynthesis route, in addition to attenuation of the pck gene.

Thus, for example, for the preparation of L-lysine

-   -   at the same time the dapA gene which codes for         dihydrodipicolinate synthase can be over-expressed (EP-B 0 197         335), or     -   at the same time a DNA fragment which imparts         S-(2-aminoethyl)-cysteine resistance can be amplified (EP-A 0         088 166).

Thus, for example, for the preparation of L-threonine

-   -   at the same time the hom gene which codes for homoserine         dehydrogenase (Peoples et al., Molecular Microbiology 2, 63-72         (1988)) or the hom^(dr) or hom_(FBR) allele which codes for a         “feed back resistant” homoserine dehydrogenase (Archer et al.,         Gene 107, 53-59 (1991); Reinscheid et al., Journal of         Bacteriology 173, 3228-3230 (1991)) can be over-expressed.

In addition to attenuation of the pck gene it may furthermore be advantageous, for the production of L-amino acids, in particular lysine and threonine, to eliminate undesirable side reactions (Nakayama: “Breeding of Amino Acid Producing Micro-organisms”, in: Overproduction of Microbial Products, Krumphanzl, Sikyta, Vanek (eds.), Academic Press, London, UK, 1982).

The microorganisms prepared according to the invention can be cultured continuously or discontinuously in the batch process (batch culture) or in the fed batch (feed process) or repeated fed batch process (repetitive feed process) for the purpose of production of L-amino acids, in particular L-lysine and L-threonine. A summary of known culture methods is described in the textbook by Chmiel (Bioprozesstechnik 1. Einführung in die Bioverfahrenstechnik [Bioprocess Technology 1. Introduction to Bioprocess Technology (Gustav Fischer Verlag, Stuttgart, 1991)) or in the textbook by Storhas (Bioreaktoren und periphere Einrichtungen [Bioreactors and Peripheral Equipment] (Vieweg Verlag, Braunschweig/Wiesbaden, 1994)).

The culture medium to be used must meet the requirements of the particular strains in a suitable manner. Descriptions of culture media for various microorganisms are contained in the handbook “Manual of Methods for General Bacteriology” of the American Society for Bacteriology (Washington D.C., USA, 1981). Sugars and carbohydrates, such as e.g. glucose, sucrose, lactose, fructose, maltose, molasses, starch and cellulose, oils and fats, such as e.g. soya oil, sunflower oil, groundnut oil and coconut fat, fatty acids, such as e.g. palmitic acid, stearic acid and linoleic acid, alcohols, such as e.g. glycerol and ethanol, and organic acids, such as e.g. acetic acid, can be used as the source of carbon. These substances can be used individually or as a mixture. Organic nitrogen-containing compounds, such as peptones, yeast extract, meat extract, malt extract, corn steep liquor, soya bean flour and urea, or inorganic compounds, such as ammonium sulphate, ammonium chloride, ammonium phosphate, ammonium carbonate and ammonium nitrate, can be used as the source of nitrogen. The sources of nitrogen can be used individually or as a mixture. Phosphoric acid, potassium dihydrogen phosphate or dipotassium hydrogen phosphate or the corresponding sodium-containing salts can be used as the source of phosphorus. The culture medium must furthermore comprise salts of metals, such as e.g. magnesium sulfate or iron sulfate, which are necessary for growth. Finally, essential growth substances, such as amino acids and vitamins, can be employed in addition to the abovementioned substances. Suitable precursors can moreover be added to the culture medium. The starting substances mentioned can be added to the culture in the form of a single batch, or can be fed in during culturing in a suitable manner.

Basic compounds, such as sodium hydroxide, potassium hydroxide, ammonia or aqueous ammonia, or acid compounds, such as phosphoric acid or sulfuric acid, can be employed in a suitable manner to control the pH. Antifoams, such as e.g. fatty acid polyglycol esters, can be employed to control the development of foam. Suitable substances having a selective action, e.g. antibiotics, can be added to the medium to maintain the stability of plasmids. To maintain aerobic conditions, oxygen or oxygen-containing gas mixtures, such as e.g. air, are introduced into the culture. The temperature of the culture is usually 20° C. to 45° C., and preferably 25° C. to 40° C. Culturing is continued until a maximum of the desired L-amino acid has formed. This target is usually reached within 10 hours to 160 hours.

The following microorganism has been deposited at the Deutsche Sammlung für Mikrorganismen und Zellkulturen (DSMZ=German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany) in accordance with the Budapest Treaty:

Escherichia coli strain DH5α/pK19mobsacBΔpck as DSM 13047

The process according to the invention is used for the fermentative preparation of L-amino acids, in particular L-aspartic acid, L-asparagine, L-homoserine, L-threonine, L-isoleucine, L-glutamate, and L-methionine, with coryneform bacteria, in particular the preparation of L-lysine, L-threonine, and L-glutamate.

EXAMPLES

The present invention is explained in more detail in the following with the aid of the following examples.

For this purpose, inter alia, experiments were carried out with the lysine producer Corynebacterium glutamicum strain MH20-22B and the threonine producer Brevibacterium flavum strain DM368-2. Strain MH20-22B has been deposited as DSM5715 (EP-B-0435 132) and strain DM368-2 as DSM5399 EP-B-0385 940) at the Deutsche Sammlung für Mikroorganismen und Zellkulturen (DSMZ, Braunschweig, Germany) in accordance with the Budapest Treaty.

Example 1

Isolation of the pck Gene

To isolate the PEP carboxykinase gene (pck) from C. glutamicum a cosmid gene library was set up on the basis of the cosmid pHC79 (Hohn and Collins, Gene 11 (1980) 291-298) by a known method (Sambrook et al., Molecular Cloning, A Laboratory Handbook, 1989, Cold Spring Harbor Laboratory Press). For this, chromosomal DNA was isolated from C. glutamicum ATCC13032 (Eikmanns et al., Microbiology 140 (1994) 1817-1828) and partly digested with the restriction enzyme Sau3A. After ligation of the resulting fragments into the BamHI cleavage site of the cosmid pHC79, the batch was packed into the protein envelope of the lambda bacteriophage and the E. coli strain ED8654 (Murray et al. Molecular and General Genetics 150 (1997) 53-61) was transfected with this. The recombinant cosmids were packed into the protein envelope of the lambda phage by a method of Sternberg et al. (Gene 1 (1979) 255-280), and the transfection of E. coli ED8654 was carried out by a method of Sambrook et al. (Molecular Cloning, A Laboratory Handbook, 1989, Cold Spring Harbor Laboratory Press). The corresponding cosmids were isolated from a total of 30 resulting recombinant E. coli clones (Sambrook et al., Molecular Cloning, A Laboratory Handbook, 1989, Cold Spring Harbor Laboratory Press) and subjected to a restriction analysis with the enzyme HindIII. It was found that 24 of the cosmids possessed inserts, and that the inserts had sizes of approximately 35 kb. A total of 2200 cosmid-carrying E. coli clones were combined, and the cosmid DNA was prepared from this mixture by known processes (Sambrook et al., Molecular Cloning, A Laboratory Handbook, 1989, Cold Spring Harbor Laboratory Press).

To isolate the pck gene from C. glutamicum, the cosmide gene library was transformed into the PEP carboxykinase-defective E. coli mutant HG4 (Goldie and Sanwal, Journal of Bacteriology 141 (1980) 115-1121) by known processes (Sambrook et al., Molecular Cloning, A Laboratory Handbook, 1989, Cold Spring Harbor Laboratory Press). Because it is PEP carboxykinase-defective, the mutant HG4 is no longer capable of growing on succinate as the only source of carbon. After transformation of the cosmid gene library into this mutant, a total of 1200 clones were obtained. Of these, a total of two clones showed growth on M9 minimal medium (Sambrook et al., Molecular Cloning, A Laboratory Handbook, 1989, Cold Spring Harbor Laboratory Press) with succinate (0.4%) as the only source of carbon. After isolation of the corresponding cosmids (Sambrook et al., Molecular Cloning, A Laboratory Handbook, 1989, Cold Spring Harbor Laboratory Press) from these clones and renewed transformation into the E. coli mutant HG4, the resulting clones were again capable of growing on M9 medium with succinate as the only source of carbon.

To limit the pck gene from C. glutamicum to a smaller fragment, the two complementing cosmids were digested with the restriction enzymes XhoI, ScaI and PvuII and separated in an electrical field on a 0.8% agarose gel by known methods (Sambrook et al., Molecular Cloning, A Laboratory Handbook, 1989, Cold Spring Harbor Laboratory Press). Fragments in the size range above 3.0 kb were isolated from the gel by electroelution (Sambrook et al., Molecular Cloning, A Laboratory Handbook, 1989, Cold Spring Harbor Laboratory Press) and ligated into the SalI (XhoI-digestion) or into the Klenow-treated EcoRI cleavage site (ScaI and PvuII digestion) of the vector pEK0 (Eikmanns et al., Gene 102 (1991) 93-98). E. coli HG4 was transformed with the ligation batches and the resulting transformants were investigated again for their ability to grow on succinate as the sole source of carbon. Seven clones with plasmids which allowed growth of the mutant HG4 on succinate were found in the transformation batch with the PvuII ligation batch. The corresponding plasmids were isolated from the recombinant strains and subjected to restriction mapping. It was found that all seven plasmids carried the same 4.3 kb PvuII insert, three in one orientation and four in the other. The newly constructed plasmids were called pEK-pckA and pEK-pckB, depending on the orientation of the insert in the vector. The restriction maps of the two plasmids are shown in FIGS. 1 and 2.

Example 2

Sequencing of the pck Structural Gene and Adjacent Regions

For the sequencing, the approximately 3.9 kb EcoRI fragment was isolated from pEK-pckA (an EcoRI cleavage site here originates from the vector pEK0) by known methods. The overhanging ends of the fragment were filled up to blunt ends with Klenow polymerase (Sambrook et al., Molecular Cloning, A Laboratory Handbook, 1989, Cold Spring Harbor Laboratory Press) and ligated into the EcoRV cleavage site of the vector pGEM-5Zf(+)(Promega Corporation, Madison, Wis., USA). The insertion of the plasmid thus generated was sequenced by the chain-stopping sequencing method (Sanger et al., Proceedings of the National Academy of Sciences USA, 74 (1977) 5463-5467). It is shown as SEQ ID No. 1. The resulting nucleotide sequence of 3935 bp was analysed with the HUSAR program package (release 3.0) of the Deutsche Krebsforschungszentrum (DKFZ=German Cancer Research Centre, Heidelberg, Germany). The sequence analysis of the fragments showed an open reading frame of 1830 bp in length, which codes for a protein consisting of 610 amino acids.

Example 3

Over-expression of the pck Gene

The plasmids pEK-pckA and pEK-pckB were introduced into the C. glutamicum strain ATCC13032 by electroporation with subsequent selection on kanamycin(50 μg/ml)-containing BHI agar plates (Liebl et al., FEMS Microbiology Letters 65 (1989) 299-304) and the resulting strains were called ATCC13032/pEK-pckA and ATCC13032/pEK-pckB. These two strains and the starting strain were cultured in Luria-Bertani complex medium [Sambrook et al., Molecular Cloning, A laboratory manual (1989) Cold Spring Harbor Laboratory Press] and the PEP carboxykinase test was carried out in accordance with the method described by Bentle and Lardy [Journal of Biological Chemistry 251 (1976) 2916-2921]. The result of the analysis is shown in table 1, and shows that the PEP carboxykinase activity in the two strains with the plasmids pEK-pckA and pEK-pckB is 10 to 12 times higher than in the starting strain.

TABLE 1 PEP carboxykinase activity in various strains PEP carboxykinase Strain (nmol min⁻¹ mg protein⁻¹) ATCC13032  120 ATCC13032/pEK-pckA 1270 ATCC13032/pEK-pckB 1510

Example 4

Preparation of an Integration Plasmid for Deletion Mutagenesis of the pck Gene

For inactivation of the PEP carboxykinase gene, the EcoRI-SacI fragment of the pck gene was isolated from the vector pEK-pckB (FIG. 2) and ligated into the vector pGEM-7Zf(+) (Promega Corporation, Madison, Wis., USA). A pck-internal 1.07 kb HindII-HindIII fragment was deleted from the resulting plasmid, and the pck gene with the 1.07 kb deletion was then isolated as the BfrI-SacI frament and, after the overhanging ends had been filled up, was ligated into the vector pk19mobsacB, which is non-replicative in C. glutamicum (Schäfer et al., Gene 145, 69-73 (1994)). In the integration plasmid pK19mobsacBΔpck (FIG. 3) constructed in this way, the 5′ region of the pck gene (350 bp) is directly adjacent to the 3′ region of the pck gene (340 bp); in the genome, the two regions are separated from one another by 1071 bp. Up to this step, all clonings were carried out in E. coli DH5α as the host.

Example 5

Deletion Mutagenesis of the pck Gene in the Lysine Producer MH20-22B

E. coli S17-1 was then transformed with the integration plasmid pK19mobsacBΔpck (Simon et al., Bio/Technology 1,784-791 (1983)). This strain allows transfer of a plasmid to Corynebacterium glutamicum by conjugation (Schäfer et al., Journal of Bacteriology 172 (1990) 1663-1666). The lysine production strain C. glutamicum MH20-22B was used as the recipient of the conjugation (Schrumpf et al., Applied Microbiology and Biotechnology 37 (1992) 566-571)). Several transconjugants were obtained from the conjugation between E. coli S17-1/pk19mobsacBΔpck and C. glutamicum MH20-22B and subsequent selection on Luria-Bertani agar plates with kanamycin (25 μg/ml) and nalidixic acid (50 μg/ml). For selection for the second recombination event, which is to lead to excision of the vector together with the pck gene, these transconjugants were cultured on antibiotic-free Luria-Bertani complex medium [Sambrook et al; Molecular Cloning, A laboratory manual (1989) Cold Spring Harbor Laboratory Press] with 1% glucose and then plated out on the same medium plus 10% sucrose. The sacB gene present on the vector pk19mobsacB codes for the enzyme levan sucrase and leads to synthesis of levan from sucrose. Since levan is toxic to C. glutamicum, only C. glutamicum cells which have lost the integration plasmid can grow on sucrose-containing medium (Jäger et al., Journal of Bacteriology 174 (1992) 5462-5466). 30 sucrose-resistant clones were investigated for their kanamycin sensitivity. For 11 of the clones tested, in addition to the sucrose resistance, the desired kanamycin sensitivity could also be confirmed. In these 11 clones, the vector background had therefore been excised again. Whether the desired deletion had also taken place was tested by analysis by means of the polymerase chain reaction (PCR). For this, chromosomal DNA was liberated from a colony of the starting strain and from colonies of the 11 kanamycin-sensitive clones. For this, the particular colony was removed from the agar plate with a toothpick, suspended in 50 μl H₂O and incubated for 5 minutes at 95° C. 1 μl portions of the resulting solution were each employed as templates in the PCR. Oligonucleotides which cover the regions from nucleotide 2136 to 2158 and from 3815 to 3793 in SEQ ID No. 1 were used as primers. The PCR conditions were: prior denaturing: 150 seconds at 94° C.; denaturing 60 seconds at 94° C.; hybridization 30 seconds at 60° C.; amplification 120 seconds at 72° C.; 30 cycles, end extension 240 seconds at 72° C. On the basis of the primers chosen, a PCR product of 1.68 kb was expected in the batch with the DNA of the starting strain. A PCR product of 0.61 kb was expected in the PCR with the pck deletion mutant. A PCR product 0.61 kb in size was obtained with one clone. The desired deletion of the internal 1071 bp pck fragment in this clone was thereby demonstrated. The clone was called MH20-22BΔpck. The 1.68 kb PCR product was detected in the batches of the other clones. In these, the vector had thus been excised such that the genomic starting situation was re-established.

The strain MH20-22BΔpck and the starting strain MH20-22B were grown in Luria-Bertani complex medium plus 1% glucose and the PEP carboxykinase test was carried out in accordance with the method described by Bentle and Lardy (Journal of Biological Chemistry 251 (1976) 2916-2921). The result of the analysis (table 2) shows that PEP carboxykinase activity is no longer detectable in the mutant MH20-22BΔpck, in contrast to the starting strain MH20-22B.

TABLE 2 PEP carboxykinase activity in various strains PEP carboxykinase Strain (nmol min⁻¹ mg protein⁻¹) MH20-22B 65 MH20-22BΔpck  <3* *3 nmol min⁻¹ mg protein⁻¹ is the detection limit

Example 6

Production of L-lysine

To investigate the effect of the inactivation of the PEP carboxykinase gene on lysine production, the strain MH20-22B (Schrumpf et al., Applied Microbiology and Biotechnology 1992, 37:566-571) and the PEP carboxykinase-negative mutant MH20-22BΔpck (example 5) were cultured in Luria-Bertani complex medium plus 1% glucose and the fermentation medium CGXII (Keilhauer et al., Journal of Bacteriology 1993, 175:5595-5603) was inoculated from the two pre-cultures (5% inokulum, optical density at 600 nm approximately 0.5). The medium additionally comprised 3 mM leucine, since the two strains are leucine-auxotrophic. The batches each comprise 60 ml culture, obtained in 500 ml conical flasks with baffles. After culture for 24 hours at 28° C. on a rotary shaking machine of the Certomat S/50 type (B. Braun Biotech International, Melsungen, Germany) at 120 rpm, the concentration of the lysine secreted into the medium was determined.

The amino acid concentration was determined by means of high pressure liquid chromatography (Jones und Gilligan, Journal of Chromatography 1983, 266:471-482). The result of the fermentation is shown in

TABLE 3 Lysine concentration in the culture supernatant of the strains MH20-22B and MH20-22BΔpck Strain L-Lysine (mM) MH20-22B 54 MH20-22BΔpck 65

Example 7

Deletion Mutagenesis of the pck Gene in the Threonine Producer DM368-2

As in the case of the lysine producer MH20-22B, using the E. coli strain S17-1/pk19mobsacBΔpck a conjugation with the threonine producer DM368-2 with subsequent selection for the first and second recombination was carried out (see example 5). Of 30 sucrose-resistant clones, 14 were kanamycin-sensitive. Of these, the 1071 bp deletion in the pck gene could be detected with the aid of the PCR analysis described in example 5 in two clones, called DM368-2Δpck16 and DM368-2Δpck18.

An enzyme test with the starting strain DM368-2 and the two pck deletion strains DM368-2Δpck16 and DM368-2Δpck18, carried out as described in example 5, showed that no PEP carboxykinase activity is detectable in these mutants (Table 4).

TABLE 4 PEP carboxykinase activity in various strains PEP carboxykinase Strain (nmol min⁻¹ mg protein⁻¹) DM368-2 79 DM368-2BΔpck16  <3* DM368-2BΔpck18  <3* *3 nmol min⁻¹ mg protein⁻¹ is the detection limit

Example 8

Production of L-threonine

Analogously to the experiments on L-lysine production, the accumulation of threonine in the culture supernatant of the PEP carboxykinase-defective strain DM368-2BΔpck16 was investigated in comparison with the starting strain DM368-2. For this, the two strains were cultured in Luria-Bertani complex medium plus 1% glucose and the fermentation medium CGXII was inoculated from the precultures. After culture for 24 hours at 28° C. on a rotary shaking machine at 120 rpm, the concentration of the threonine secreted into the medium was determined.

The amino acid concentration was determined by means of high pressure liquid chromatography (see above). The result of the fermentation is shown in table 5.

TABLE 5 Threonine concentration in the culture supernatant of the strains DM 368-2 and DM 368-2Δpck16 Strain L-Threonine (mM) DM368-2  8 DM368-2Δpck16 22

Example 9

Deletion Mutagenesis of the pck Gene in C. glutamicum Wild-type Strain ATCC13032

A conjugation with the C. gutamicum wild type-strain ATCC13032 was carried out with the E. coli strain S 17-1/pK19 mobsacBΔpck (example 4) as in the case of the lysine producer MH20-22B (example 5) and of the threonine producer DM368-2 (example 7). The selection on the first and second recombination event was carried out as in example 5. Of 52 saccharose-resistant clones, 8 were kanamycin-sensitive. Of these, the 1071 bp deletion in the pck gene was able to be demonstrated with the aid of the FCR analysis described in example 5 in three clones designated as ATCC13032Δpck2, ATCC13032Δpck29 and ATCC13032Δpck30.

An enzyme test with the initial strain ATCC13032 and two of the three pck deletion strains, ATCC13032Δpck2 and ATCC13032Δpck29, carried out as described as in example 5, showed that no PEP carboxkinase activity can be demonstrated in both mutants (table 6).

TABLE 6 PEP carboxykinase activity In different strains Strain PEP carboxykinase ATCC13032 132  ATCC13032Δpck2 <3* ATCC13032Δpck29 <3* *3 nmol min⁻¹ mg protein⁻¹ is the demonstration limit

Example 10

Production of L-glutamate

The accumulation of L-glutamate in the culture supernatant of the PEP carboxykinase-defective strains ATCC13032Δpck2 and ATCC13032Δpck29 in comparison to the initial strain ATCC13032 was also investigated in analogy with the experiments on the production of L-lysine and of L-threoninc (examples 6, 8). To this end the three strains were cultivated in Luria-Bertani complex [complexing] medium plus 1% glucose and the fermentation medium CGXII (see example 6) from these precultures inoculated. 15 mg Tween 60 per ml were added to the medium 4 hours after the inoculation for the induction of the production of L-glutamate. After cultivation for 24 hours at 30° C. on a rotary agitator at 120 rmp the concentration of the glutamate excreted into the medium was determined.

The determination of the concentration of amino acid took place by means of high-pressure liquid chromatography (see example 6).

The result of the fermentation is shown in table 7, in which the indicated values represent average values from two experiments in each instance. It turns out that the inactivation of the PEP carboxykinase gene results in a 4 to 5-fold increase of the concentration of glutamate in the medium.

TABLE 7 Concentration of glutamate in the culture supernatant of the strains ATCC13032, ATCC13032Δpck2 and ATCC13032Δpck29 Strain L-glutamate (mM) ATCC13032 11.24 ATCC13032Δpck2 49.45 ATCC13032Δpck29 52.17

In the Figures, the abbreviations and designations that are used have the following meanings:

sacB: sacB gene ori V: Replication origin V ori T: Replication origin for the transfer Km-r: Kanamycin resistance KpnI: Cleavage site of the restriction enzyme KpnI HindIII: Cleavage site of the restriction enzyme HindIII HindII: Cleavage site of the restriction enzyme HindII PstI: Cleavage site of the restriction enzyme PstI SphI: Cleavage site of the restriction enzyme SphI XbaI: Cleavage site of the restriction enzyme XbaI SalI: Cleavage site of the restriction enzyme SalI SacI: Cleavage site of the restriction enzyme SacI BfrI: Cleavage site of the restriction enzyme BfrI ScaI: Cleavage site of the restriction enzyme ScaI BamHI: Cleavage site of the restriction enzyme BamHI EcoRI: Cleavage site of the restriction enzyme EcoRI pck′: 3′ terminal fragment of the pck gene pck″: 5′ terminal fragment of the pck gene pck: pck gene 

1. A process for the fermentative preparation of L-amino acids selected from the group consisting of L-lysine, L-threonine, and L-glutamate in Corynebacterium comprising: a) growing said Corynebacterium in which DNA encoding the polypeptide of SEQ ID NO:2 is attenuated by a method of method of mutagenesis selected from the group consisting of insertion mutagenesis by insertion of at least one base pair, deletion mutagenesis with deletion of at least one base pair, and transition or transversion mutagenesis with incorporation of a non-sense mutation or the activity of said polypeptide is reduced as compared to an unattenuated Corynebacterium; b) concentrating the desired L-amino acid product in the medium or cells of said bacteria; and c) isolating said L-amino acid product.
 2. The process of claim 1, wherein said bacteria are of the species Corynebacterium glutamicum.
 3. The process of claim 1, wherein said polynucleotide is attenuated by integration mutagenesis using plasmid pkl9mobsacBΔpck, as shown in FIG. 3 and deposited as DSM
 13047. 4. The process of claim 1, wherein said amino acid product is L-lysine and said bacteria over-express the dapA gene which codes for dihydrodipicolinate synthase.
 5. The process of claim 1, wherein said amino acid product is L-lysine and wherein said bacteria contain a DNA fragment encoding S-(2-aminoethyl)-cysteine resistance wherein both have been amplified.
 6. The process of claim 1, wherein said amino acid product is L-threonine and wherein said bacteria over-express the hom gene which codes for homoserine dehydrogenase.
 7. The process of claim 1, wherein said amino acid product is L-threonine and wherein said bacteria over-express the gene that codes for either hom^(dr) or hom^(fbr).
 8. The process of claim 1, wherein said amino acid product is L-glutamate.
 9. The process of claim 1, wherein said L-amino acid is L-lysine.
 10. The process of claim 1, wherein said L-amino acid is L-threonine.
 11. The process of claim 1, wherein said L-amino acid is L-glutamate.
 12. The process of claim 2, wherein said bacteria are of the species Corynebacterium glutamicum.
 13. The process of claim 1, wherein said DNA is attenuated by a method of mutagenesis selected from the group consisting of: insertion mutagenesis with insertion of at least one base pair, deletion mutagenesis with deletion of at least one base pair and transition or transversion mutagenesis with incorporation of a non-sense mutation.
 14. A process for the fermentative preparation of L-amino acids selected from the group consisting of L-lysine, L-threonine and L-glutamate in Corynebacterium, comprising: (a) growing said Corynebacterium in which DNA encoding a polypeptide having phosphoenol pyruvate carboxykinase activity and comprising the amino acid sequence of SEQ ID NO: 2 is attenuated by a method of mutagenesis selected from the group consisting of insertion mutagenesis by insertion of at least one base pair, deletion mutagenesis with deletion of at least one base pair, and transition or transversion mutagenesis with incorporation of a non-sense mutation or the activity of said polypeptide is reduced as compared to an unattenuated Corynebacterium: (b) concentrating the desired L-amino acid product in the medium or cells of said Corynebacterium; and (c) isolating said L-amino acid product. 